The cytosolic fraction from 3T3 cells of Balb/c mouse fibroblast showed weak nucleotide sugar pyrophosphatase activity with 5-fluorouridine 5’-diphosphate a-D-galactose (FUDPGal). Unlike the membrane fraction from 3T3 cells, which exhibited no such activity with FUDPGal or 5-fluorouridine 5’-diphosphate N-acetyl- a-D-glucosamine (FUDPGlcNAc), the membrane fraction from simian virus 40-transformed 3T3 (3T3SV) cells showed higher hydrolytic activities with these nucleotide sugar analogs than the cytosolic fraction. Both FUDPGal and FUDPGlcNAc exhibited potent cytotoxic effects comparable to that of 5-fluorouridine (FUR) on 3T3SV cells but did not affect the viability of the native 3T3 cells even at 1 mM. FUR was detected only in the culture of 3T3SV cells grown with FUDPGal or FUDPGlcNAc. These results revealed the critical role of membrane-associated nucleotide sugar pyrophosphatase in the selective cytotoxic effects of FUDPGal and FUDPGlcNAc.